Secretory protein expression is the expression of a protein in a host cell, where the protein is exported to the cell membrane and is either solubly released into the medium or remains attached to the cell membrane. Secretory protein expression is mediated by a signal peptide at the N-terminus of the protein which directs the polypeptide to the membrane.
Usually, recombinant proteins that are produced in prokaryotic hosts such as E. coli are produced intracellularly. When the protein is recovered in such a procedure, the cells have to be lysed which leads to contamination of the recombinant protein with cellular content. The protein then has to be recovered from whole cell extracts in multi-step purification procedures, which is time consuming and results in poor yields.
Secretion of recombinant proteins into the medium is a better strategy because purification of proteins from spent medium is easier and more compatible with continuous culturing. However, the present systems do not have efficient yields.
Secretory protein expression where the protein remains attached to the cell surface has other uses. Examples of use for this type of protein expression include live-vaccine development, epitope mapping, biosorbent and biosensor development and the high throughput screening of protein and peptide libraries for drug discovery.
In both surface display and secretion, recombinant proteins face the challenge of translocation across the complex E. coli cell envelope that consists of two lipid membranes (the inner and outer membrane) with a gel-like compartment, the periplasm, in between. This has been shown to be very difficult and the methods previously used have had low efficacy.
Autotranporters are large proteins that are secreted by Gram-negative bacteria, such as E. coli. The autotransporter system is simple in the sense that the autotransporter, as implied by its name, is suggested to carry all information for translocation across the periplasm and outer membrane within the protein itself. However, the mechanism whereby autotransporters are secreted is still not completely understood.
Autotransporters are synthesized as large precursor proteins that contain three main domains: (i) an N-terminal signal peptide that targets the protein to the Sec translocon and initiates transfer across the inner membrane, (ii) a passenger domain which comprises the “cargo” protein that is to be secreted and (iii) a C-terminal pore-forming domain (translocator domain) comprising a beta barrel structure that integrates into the outer membrane and plays a crucial but unclear role in translocation of the passenger domain across the outer membrane into extracellular space.
After translocation, the passenger domain is cleaved from the translocator domain and is released into the extracellular environment. In some cases, the passenger domain remains non-covalently attached to the cell surface. Cleavage can be achieved by the action of an (external) protease on a protease motif situated between the translocator domain and the passenger domain. Alternatively, cleavage takes place through an intramolecular autocatalytic event at a specific site between the translocator domain and the passenger domain.
The passenger domain of an autotransporter comprises a beta stem structure and side domains. The beta stem is an elongated structure formed by an extended beta helix. The C-terminus of the passenger domain comprises an autochaperone domain which has been implicated in both passenger folding and translocation across the outer membrane.
Hbp is an autotransporter protein that belongs to the subfamily of serine protease autotransporters of Enterobacteriaceae (SPATEs). The crystal structure of the passenger domain of Hbp has recently been determined (Otto et al. 2005 J Biol Chem 280(17): 17339-45), and is shown as FIG. 11A. The structure shows that the polypeptide forms a long right-handed beta-helical structure (“beta stem”). The passenger domain of the Hbp comprises two larger side domains, domain d1 and domain d2, of which d1 comprises the serine proteinase activity of the protein and d2 has an unknown function. There are also three smaller side domains, domain 3 (d3), domain 4 (d4) and domain 5 (d5).
Similar beta stem domains have been shown also for other autotransporters such as pertactin (Emsley et al 1996 Nature 381: 90-92) and IgA protease (Johnson et al 2009 J Mol Biol 389(3): 559-74).
There have been previous attempts in using autotransporters for secretory protein expression in E. coli, mostly using variants of the Neisserial IgA protease (Pyo et al 2009 Vaccine 27 2030-2036) and the endogenous E. coli autotransporter AIDA-I (Van Gerven et al 2009 Microbiology 155:468-476) that were engineered for surface display purposes.
Efforts using IgA protease and AIDA-I for secretion of recombinant proteins used constructs which resulted in poor yields of secreted and surface exposed protein (Pyo et al 2009 Vaccine 27 2030-2036; Van Gerven et al 2009 Microbiology 155:468-476). In the majority of such studies the complete, or almost complete, endogenous passenger domain was replaced by the recombinant protein.
So far, autotransporters have mainly been used as a display platform rather than for secretion of heterologous proteins in soluble form, where the protein is secreted into the medium.
IgA protease requires an accessory protease for processing whereas AIDA-I remains non-covalently attached to the outer membrane after cleavage. Thus, these autotransporters can only be used for surface presentation of epitopes and proteins.
Efficient display and secretion of calmodulin fused the passenger of Hbp has previously been shown (Jong et al 2007 Molecular Microbiology 63:1524-1536). In order to minimize perturbation of the native β-stem of the passenger, calmodulin replaced domain 2 of the Hbp passenger.
For certain applications the possibility to secrete or display more than one protein of interest (POI) from/on the cell surface is very useful. Such applications include vaccines, for example in which two or more epitopes are displayed on the same cell surface, enzyme display, in which more than one enzyme is displayed on the cell surface in order to carry out a range of catalytical reactions in a series of steps, exposure of peptide libraries and inhibitor screening.
For multivalent vaccines it is particularly useful to have a system wherein one population of host cells can express and display or secrete multiple antigens, rather than having a mixture of cell populations, each displaying or secreting only one of the antigens. Having only one cell population displaying or secreting multiple antigens has the advantage of easier production and better control of the vaccine content.
In conclusion, there is a need for improved secretory expressions systems for the display of heterologous proteins as well as secretion of heterologous proteins in soluble form into the culture medium. There is also a need for a system and a method that enable secretory protein expression of more than one protein of interest on the cell surface of a host cell or secretion of more than one protein into the culture medium.